Active salt forms with tyrosine kinase activity

ABSTRACT

The present invention relates to orally active salt forms of the mesylate salt of 4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylic acid methylamide which inhibit, regulate and/or modulate tyrosine kinase signal transduction, compositions which contain these compounds, and methods of using them to treat tyrosine kinase-dependent diseases and conditions, such as angiogenesis, cancer, tumor growth, atherosclerosis, age related macular degeneration, diabetic retinopathy, retinal ischemia, macular edema, inflammatory diseases, and the like in mammals.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to orally active salt forms of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide, which inhibit, regulate and/or modulate tyrosinekinase signal transduction, compositions which contain these salts, andmethods of using them to treat tyrosine kinase-dependent diseases andconditions, such as angiogenesis, cancer, tumor growth, atherosclerosis,age related macular degeneration, diabetic retinopathy, retinalischemia, macular edema, inflammatory diseases, and the like in mammals.

[0002] Tyrosine kinases-are a class of enzymes that catalyze thetransfer of the terminal phosphate of adenosine triphosphate to tyrosineresidues in protein substrates. Tyrosine kinases play critical roles insignal transduction for a number of cell functions via substratephosphorylation. Though the exact mechanisms of signal transduction isstill unclear, tyrosine kinases have been shown to be importantcontributing factors in cell proliferation, carcinogenesis and celldifferentiation.

[0003] Tyrosine kinases can be categorized as receptor type ornon-receptor type. Receptor type tyrosine kinases have an extracellular,a transmembrane, and an intracellular portion, while non-receptor typetyrosine kinases are wholly intracellular.

[0004] The receptor-type tyrosine kinases are comprised of a largenumber of transmembrane receptors with diverse biological activity. Infact, about twenty different subfamilies of receptor-type tyrosinekinases have been identified. One tyrosine kinase subfamily, designatedthe HER subfamily, is comprised of EGFR, HER2, HER3, and HER4. Ligandsof this subfamily of receptors include epithileal growth factor, TGF-α,amphiregulin, HB-EGF, betacellulin and heregulin. Another subfamily ofthese receptor-type tyrosine kinases is the insulin subfamily, whichincludes INS-R, IGF-IR, and IR-R. The PDGF subfamily includes the PDGF-αand β receptors, CSFIR, c-kit and FLK-II. Then there is the FLK familywhich is comprised of the kinase insert domain receptor (KDR), fetalliver kinase 1 (FLK-1), fetal liver kinase-4 (FLK-4) and the fms-liketyrosine kinase-1 (flt-1). The PDGF and FLK families are usuallyconsidered together due to the similarities of the two groups. For adetailed discussion of the receptor-type tyrosine kinases, see Plowmanet al., DN&P 7(6):334-339, 1994, which is hereby incorporated byreference.

[0005] The non-receptor type of tyrosine kinases is also comprised ofnumerous subfamilies, including Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps,Fak, Jak, Ack, and LIMK. Each of these subfamilies is furthersub-divided into varying receptors. For example, the Src subfamily isone of the largest and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr,and Yrk. The Src subfamily of enzymes has been linked to oncogenesis.For a more detailed discussion of the non-receptor type of tyrosinekinases, see Bolen Oncogene, 8:2025-2031 (1993), which is herebyincorporated by reference.

[0006] Both receptor-type and non-receptor type tyrosine kinases areimplicated in cellular signaling pathways leading to numerous pathogenicconditions, including cancer, psoriasis and hyperimmune responses.

[0007] Several receptor-type tyrosine kinases, and the growth factorsthat bind thereto, have been suggested to play a role in angiogenesis,although some may promote angiogenesis indirectly (Mustonen and Alitalo,J. Cell Biol. 129:895-898, 1995). One such receptor-type tyrosine kinaseis fetal liver kinase 1 or FLK-1. The human analog of FLK-1 is thekinase insert domain-containing receptor KDR, which is also known asvascular endothelial cell growth factor receptor 2 or VEGFR-2, since itbinds VEGF with high affinity. Finally, the murine version of thisreceptor has also been called NYK (Oelrichs et al., Oncogene 8(1):11-15,1993). VEGF and KDR are a ligand-receptor pair that play an importantrole in the proliferation of vascular endothelial cells, and theformation and sprouting of blood vessels, termed vasculogenesis andangiogenesis, respectively.

[0008] Angiogenesis is characterized by excessive activity of vascularendothelial growth factor (VEGF). VEGF is actually comprised of a familyof ligands (Klagsburn and D=Amore, Cytokine & Growth Factor Reviews7:259-270, 1996). VEGF binds the high affinity membrane-spanningtyrosine kinase receptor KDR and the related fms-like tyrosine kinase-1,also known as Flt-1 or vascular endothelial cell growth factor receptor1 (VEGFR-1). Cell culture and gene knockout experiments indicate thateach receptor contributes to different aspects of angiogenesis. KDRmediates the mitogenic function of VEGF whereas Flt-1 appears tomodulate non-mitogenic functions such as those associated with cellularadhesion. Inhibiting KDR thus modulates the level of mitogenic VEGFactivity. In fact, tumor growth has been shown to be susceptible to theantiangiogenic effects of VEGF receptor antagonists. (Kim et al., Nature362, pp. 841-844, 1993).

[0009] Solid tumors can therefore be treated by tyrosine kinaseinhibitors since these tumors depend on angiogenesis for the formationof the blood vessels necessary to support their growth. These solidtumors include histiocytic lymphoma, cancers of the brain, genitourinarytract, lymphatic system, stomach, larynx and lung, including lungadenocarcinoma and small cell lung cancer. Additional examples includecancers in which overexpression or activation of Raf-activatingoncogenes (e.g., K-ras, erb-B) is observed. Such cancers includepancreatic and breast carcinoma. Accordingly, inhibitors of thesetyrosine kinases are useful for the prevention and treatment ofproliferative diseases dependent on these enzymes.

[0010] The angiogenic activity of VEGF is not limited to tumors. VEGFaccounts for most of the angiogenic activity produced in or near theretina in diabetic retinopathy. This vascular growth in the retina leadsto visual degeneration culminating in blindness. Ocular VEGF mRNA andprotein are elevated by conditions such as retinal vein occlusion inprimates and decreased pO₂ levels in mice that lead toneovascularization. Intraocular injections of anti-VEGF monoclonalantibodies or VEGF receptor immunofusions inhibit ocularneovascularization in both primate and rodent models. Regardless of thecause of induction of VEGF in human diabetic retinopathy, inhibition ofocular VEGF is useful in treating the disease.

[0011] Expression of VEGF is also significantly increased in hypoxicregions of animal and human tumors adjacent to areas of necrosis. VEGFis also upregulated by the expression of the oncogenes ras, raf, src andmutant p53 (all of which are relevant to targeting cancer). Monoclonalanti-VEGF antibodies inhibit the growth of human tumors in nude mice.Although these same tumor cells continue to express VEGF in culture, theantibodies do not diminish their mitotic rate. Thus tumor-derived VEGFdoes not function as an autocrine mitogenic factor. Therefore, VEGFcontributes to tumor growth in vivo by promoting angiogenesis throughits paracrine vascular endothelial cell chemotactic and mitogenicactivities. These monoclonal antibodies also inhibit the growth oftypically less well vascularized human colon cancers in athymic mice anddecrease the number of tumors arising from inoculated cells.

[0012] Viral expression of a VEGF-binding construct of Flk-1, Flt-1, themouse KDR receptor homologue, truncated to eliminate the cytoplasmictyrosine kinase domains but retaining a membrane anchor, virtuallyabolishes the growth of a transplantable glioblastoma in mice presumablyby the dominant negative mechanism of heterodimer formation withmembrane spanning endothelial cell VEGF receptors. Embryonic stem cells,which normally grow as solid tumors in nude mice, do not producedetectable tumors if both VEGF alleles are knocked out. Taken together,these data indicate the role of VEGF in the growth of solid tumors.Inhibition of KDR or Flt-1 is implicated in pathological angiogenesis,and these receptors are useful in the treatment of diseases in whichangiogenesis is part of the overall pathology, e.g., inflammation,diabetic retinal vascularization, as well as various forms of cancersince tumor growth is known to be dependent on angiogenesis. (Weidner etal., N. Engl. J. Med., 324, pp. 1-8, 1991).

[0013] Although similar piperazinyl compounds have been previouslyreported to be useful as tyrosine kinase inhibitors (see WO 01/17995,published Mar. 15, 2001) a need still exists for forms of the compoundsthat can be readily administered to patients, especially orally active,soluble forms of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide that have thermal stability upon storage. Accordingly,the identification of salt forms of 4-[2(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide, which specifically inhibit, regulate and/or modulatethe signal transduction of tyrosine kinases, is desirable and is anobject of this invention.

SUMMARY OF THE INVENTION

[0014] The present invention relates to mesylate salt forms of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide that are capable of inhibiting, modulating and/orregulating signal transduction of both receptor-type and non-receptorstype tyrosine kinases. One embodiment of the present invention isillustrated by a salt of Formula I:

[0015] wherein

[0016] A⁻ is mesylate salt.

DESCRIPTION OF THE FIGURES

[0017]FIG. 1: X-ray powder diffraction pattern of the mesylate salt(Form A) of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methyl amide.

[0018]FIG. 2: X-ray powder diffraction pattern of the mesylate salt(Form B) of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methyl amide.

DETAILED DESCRIPTION OF THE INVENTION

[0019]4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide (Compound 4-4) is an inhibitor of tyrosine kinasesignal transduction and, in particular, inhibits the kinase KDR. Thebasic piperazine nitrogen of Compound 4-4 will form salts upon treatmentwith various acids. Two polymorphic forms of the mesylate salt have beenobserved. Form A is the monohydrate and Form B is the dehydrate, oranhydrous, form.

[0020] An embodiment of the invention is illustrated by a mesylate salt(Form A) of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide, also described as1-({2-[(5-cyano-1,3-thiazol-2-yl)amino]pyridin-4-yl}methyl)-4-[(methylamino)carbonyl]piperazin-1-ium methanesulfonate, incrystalline form characterized by an X-ray powder diffraction patternhaving diffraction angles of: (with an experimental error of about±0.8⁰) 10.67, 12.28, 17.88, 18.45, 21.21, 21.49, 22.68, 23.22, 23.37,23.94, 27.12, 28.72, and 31.68.

[0021] And yet a further embodiment is the mesylate salt (Form A) of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide in crystalline form characterized by an X-ray powderdiffraction pattern having multiple diffraction peaks between 5° and 35°2-theta and a melting endotherm of 233° C. at a rate of 5° C. perminute.

[0022] An embodiment of the invention is illustrated by a mesylate salt(Form B) of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide, also described as1-({2-[(5-cyano-1,3-thiazol-2-yl)amino]pyridin-.4-yl}methyl)-4-[(methylamino)carbonyl]piperazin-1-ium methanesulfonate,in crystalline form characterized by an X-ray powder diffraction patternhaving diffraction angles of: (with an experimental error of about±0.8°) 9.62, 13.61, 15.15, 15.69, 16.33, 16.58, 17.75, 18.07, 19.37,19.62, 21.01, 21.23, 22.07, 22.35, 22.67, 22.87, 23.83, 25.05, 25.23,27.63, and 30.47.

[0023] And yet a further embodiment is the mesylate salt (Form B) of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide in crystalline form characterized by an X-ray powderdiffraction pattern having multiple diffraction peaks between 5° and 35°2-theta and a melting endotherm of 232° C. at a rate of 5° C. perminute.

[0024] The salts of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described in: E.L. Eliel and S. H.Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York,1994, pages 1119-1190), and occur as racemates, racemic mixtures, and asindividual diastereomers, with all possible isomers and mixturesthereof, including optical isomers, being included in the presentinvention. In addition, the salts disclosed herein may exist astautomers and both tautomeric forms are intended to be encompassed bythe scope of the invention, even though only one tautomeric structure isdepicted. For example, any claim to compound A below is understood toinclude tautomeric structure B, and vice versa, as well as mixturesthereof.

[0025] The instantly disclosed salts are inhibitors of tyrosine kinaseand are therefore useful to treat or prevent tyrosine kinase-dependentdiseases or conditions in 20 mammals.

[0026] “Tyrosine kinase-dependent diseases or conditions” refers topathologic conditions that depend on the activity of one or moretyrosine kinases. Tyrosine kinases either directly or indirectlyparticipate in the signal transduction pathways of a variety of cellularactivities including proliferation, adhesion and migration, anddifferentiation. Diseases associated with tyrosine kinase activitiesinclude the proliferation of tumor cells, the pathologicneovascularization that supports solid tumor growth, ocularneovascularization (diabetic retinopathy, age-related maculardegeneration, retinal ischemia, macular edema, and the like) andinflammation (psoriasis, rheumatoid arthritis, and the like). Intreating such conditions with the instantly claimed salts, the requiredtherapeutic amount will vary according to the specific disease and isreadily ascertainable by those skilled in the art. Although bothtreatment and prevention are contemplated by the scope of the invention,the treatment of these conditions is the preferred use.

[0027] Also included within the scope of the claims is a compositionwhich is comprised of a salt of the present invention and apharmaceutically acceptable carrier.

[0028] The present invention also encompasses a method of treating orpreventing cancer in a mammal in need of such treatment which iscomprised of administering to said mammal a therapeutically effectiveamount of a presently disclosed salt. The term “treating cancer” or“treatment of cancer” refers to administration to a mammal afflictedwith a cancerous condition and refers to an effect that alleviates thecancerous condition by killing the cancerous cells, but also to aneffect that results in the inhibition of growth and/or metastasis of thecancer.

[0029] Preferred cancers for treatment are selected from cancers of thebrain, genitourinary tract, lymphatic system, stomach, larynx and lung.Another set of preferred forms of cancer are histiocytic lymphoma, lungadenocarcinoma, small cell lung cancers, pancreatic cancer, gioblastomasand breast carcinoma. The utility of angiogenesis inhibitors in thetreatment of cancer is known in the literature, see J. Rak et al. CancerResearch, 55:4575-4580, 1995, for example. The role of angiogenesis incancer has been shown in numerous types of cancer and tissues: breastcarcinoma (G. Gasparini and A. L. Harris, J. Clin. Oncol., 1995,13:765-782; M. Toi et al., Japan. J. Cancer Res., 1994, 85:1045-1049);bladder carcinomas (A. J. Dickinson et al., Br. J. Urol., 1994,74:762-766); colon carcinomas (L. M. Ellis et al., Surgery, 1996,120(5):871-878); and oral cavity tumors (J. K. Williams et al., Am. J.Surg., 1994, 168:373-380).

[0030] Also included is a method of treating or preventing a disease inwhich angiogenesis is implicated, which is comprised of administering toa mammal in need of such treatment a therapeutically effective amount ofa salt of the instant invention. Such a disease in which angiogenesis isimplicated is ocular diseases such as—retinal vascularization, diabeticretinopathy, age-related macular degeneration, retinal ischemia, macularedema, and the like.

[0031] Tumors which have undergone neovascularization show an increasedpotential for metastasis. VEGF released from cancer cells enhancesmetastasis possibly by increasing extravasation at points of adhesion tovascular endothelium. (A. Amirkhosravi et al., Platelets, 10:285-292(1999).) In fact, angiogenesis-is essential for tumor growth andmetastasis. (S. P. Gunningham, et al., Can. Research, 61: 3206-3211(2001)). The angiogenesis inhibitors disclosed in the presentapplication are therefore also useful to prevent or decrease tumor cellmetastasis. Such a use is also contemplated to be within the scope ofthe present invention.

[0032] Further included within the scope of the invention is a method oftreating or preventing a disease in which angiogenesis is implicated,which is comprised of administering to a mammal in need of suchtreatment a therapeutically effective amount of a salt of the instantinvention. Ocular neovascular diseases are an example of conditionswhere much of the resulting tissue damage can be attributed to aberrantinfiltration of blood vessels in the eye. (see WO 00/30651, publishedJun. 2, 2000.) The undesirable infiltration can be triggered by ischemicretinopathy, such as that resulting from diabetic retinopathy,retinopathy of prematurity, retinal vein occlusions, etc., or bydegenerative diseases, such as the choroidal neovascularization observedin age-related macular degeneration. Inhibiting the growth of bloodvessels by administration of a salt of the instant invention wouldtherefore prevent the infiltration of blood vessels and prevent or treatdiseases where angiogenesis is implicated, such as ocular diseases likeretinal vascularization, diabetic retinopathy, age-related maculardegeneration, retinal ischemia, macular edema, and the like.

[0033] A method of treating or preventing preeclampsia is also withinthe scope of the present invention, which comprises administering atherapeutically effective amount of a salt of the instant invention.Studies have shown that the action of VEGF on the Flt-1 receptor ispivotal in the pathogenesis of preeclampsia. (Laboratory Investigation79:1101-1111 (September 1999)). Vessels of pregnant women incubated withVEGF exhibit a reduction in endothelium-dependent relaxation similar tothat induced by plasma from women with preeclampsia. In the presence ofan anti-Flt-1 receptor antibody, however, neither VEGF or plasma fromwomen with preeclampsia reduced the endothelium-dependent relaxation.Therefore the claimed salt of the instant invention serve to treatpreeclampsia via their action on the tyrosine kinase domain of the Flt-1receptor.

[0034] Also within the scope of the invention is a method of reducing orpreventing tissue damage following a cerebral ischemic event whichcomprises administering a therapeutically effective amount of a salt ofthe instant invention. The claimed salt can also be used to reduce orprevent tissue damage which occurs after cerebral ischemic events, suchas stroke, by reducing cerebral edema, tissue damage, and reperfusioninjury following ischemia. (Drug News Perspect 11:265-270 (1998); J.Clin. Invest. 104:1613-1620 (1999); Nature Med 7:222-227 (2001)).

[0035] Also included within the scope of the present invention is amethod of treating or preventing inflammatory diseases which comprisesadministering to a mammal in need of such treatment a therapeuticallyeffective amount of a salt of the instant invention. Examples of suchinflammatory diseases are rheumatoid arthritis, psoriasis, contactdermatitis, delayed hypersensitivity reactions, and the like. (A.Giatromanolaki et al., J. Pathol. 2001; 194:101-108.)

[0036] Also included is a method of treating or preventing a tyrosinekinase-dependent disease or condition in a mammal which comprisesadministering to a mammalian patient in need of such treatment atherapeutically effective amount of a salt of the instant invention. Thetherapeutic amount varies according to the specific disease and isdiscernable to the skilled artisan without undue experimentation.

[0037] A method of treating or preventing retinal vascularization whichis comprised of administering to a mammal in need of such treatment atherapeutically effective amount of a salt of the instant invention isalso encompassed by the present invention. Methods of treating orpreventing ocular diseases, such as diabetic retinopathy, age-relatedmacular degeneration, retinal ischemia and macular edema are also partof the invention. Also included within the scope of the presentinvention is a method of treating or preventing inflammatory diseases,such as rheumatoid arthritis, psoriasis, contact dermatitis and delayedhypersensitivity reactions, as well as treatment or prevention of boneassociated pathologies selected from osteosarcoma, osteoarthritis, andrickets, also known as oncogenic osteomalacia. (Hasegawa et al.,Skeletal Radiol., 28, pp. 41-45, 1999; Gerber et al., Nature Medicine,Vol. 5, No. 6, pp. 623-628, June 1999). And since VEGF directly promotesosteoclastic bone resorption through KDR/Flk-1 expressed in matureosteoclasts (FEBS Let. 473:161-164 (2000); Endocrinology, 141:1667(2000)), the instant salt forms are also useful to treat and preventconditions related to bone resorption, such as osteoporosis and Paget'sdisease.

[0038] The invention also contemplates the use of the instantly claimedsalt forms in combination with another compound selected from:

[0039] 1) an estrogen receptor modulator,

[0040] 2) an androgen receptor modulator,

[0041] 3) retinoid receptor modulator,

[0042] 4) a cytotoxic agent,

[0043] 5) an antiproliferative agent,

[0044] 6) a prenyl-protein transferase inhibitor,

[0045] 7) an HMG-CoA reductase inhibitor,

[0046] 8) an HIV protease inhibitor,

[0047] 9) a reverse transcriptase inhibitor, and

[0048] 10) another angiogenesis inhibitor.

[0049] Preferred angiogenesis inhibitors are selected from the groupconsisting of a tyrosine kinase inhibitor, an inhibitor ofepidermal-derived growth factor, an inhibitor of fibroblast-derivedgrowth factor, an inhibitor of platelet derived growth factor, an MMP(matrix metalloprotease) inhibitor, an integrin blocker, interferon-α,interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor,carboxyamidotriazole, combreta-statin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, and an antibody to VEGF. Preferred estrogen receptormodulators are tamoxifen and raloxifene.

[0050] Also included in the scope of the claims is a method of treatingcancer which comprises administering a therapeutically effective amountof a salt of the instant invention in combination with radiation therapyand/or in combination with a compound selected from:

[0051] 1) an estrogen receptor modulator,

[0052] 2) an androgen receptor modulator,

[0053] 3) retinoid receptor modulator,

[0054] 4) a cytotoxic agent,

[0055] 5) an antiproliferative agent,

[0056] 6) a prenyl-protein transferase inhibitor,

[0057] 7) an HMG-CoA reductase inhibitor,

[0058] 8) an HIV protease inhibitor,

[0059] 9) a reverse transcriptase inhibitor, and

[0060] 10) another angiogenesis inhibitor.

[0061] And yet another embodiment of the invention is a method oftreating cancer which comprises administering a therapeuticallyeffective amount of a salt of the instant invention in combination withpaclitaxel or trastuzumab.

[0062] Also within the scope of the invention is a method of reducing orpreventing tissue damage following a cerebral ischemic event whichcomprises administering a therapeutically effective amount of a salt ofthe instant invention.

[0063] Some of the abbreviations that may be used in the description ofthe chemistry and in the Examples include: ACN Acetonitrile; Ac₂O Aceticanhydride; AcOH Acetic acid; AIBN 2,2′-Azobisisobutyronitrile; BINAP2,2′-Bis(diphenylphosphino)-1,1′ binaphthyl; Bn Benzyl; BOC/Boctert-Butoxycarbonyl; BSA Bovine Serum Albumin; CAN Ceric AmmoniaNitrate; CBz Carbobenzyloxy; CI Chemical Ionization; DBAdibenzanthracene; DBAD Di-tert-butyl azodicarboxylate; DBU1,8-Diazabicyclo[5.4.0]undec-7-ene; DCE 1,2-Dichloroethane; DEADdiethylazodicarboxylate; DEM diethoxymethane; DIADdiisopropylazodicarboxylate; DIEA N,N-Diisopropylethylamine; DMACN,N-dimethylacetamide; DMAP 4-Dimethylaminopyridine; DME1,2-Dimethoxyethane; DMF N,N-Dimethylformamide; DMPU1,3-Dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone; DMSO Methylsulfoxide; DPAD dipiperidineazodicarbonyl; DPPA Diphenylphosphorylazide; DTT Dithiothreitol; EDC 1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide-hydrochloride; EDTA Ethylenediaminetetraaceticacid; ES Electrospray; ESI Electrospray ionization; Et₂O Diethyl ether;Et₃N Triethylamine; EtOAc Ethyl acetate; EtOH Ethanol; FAB Fast atombombardment; HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid;HOAc Acetic acid; HMTA Hexamethylenetetramine; HOBT1-Hydroxybenzotriazole hydrate; HOOBT3-Hydroxy-1,2,2-benzotriazin-4(3H)-one; HPLC High-performance liquidchromatography; HRMS High Resolution Mass Spectroscopy; KOtBu Potassiumtert-butoxide; LAH Lithium aluminum hydride; LCMS Liquid ChromatographyMass Spectroscopy; MCPBA m-Chloroperoxybenzoic acid; Me Methyl; MEKMethyl ethyl ketone; MeOH Methanol; MIBK Methyl isobutyl ketone; MsMethanesulfonyl; MS Mass Spectroscopy; MsCl Methanesulfonyl chloride;MsOH methanesulfonic acid; MTBE tert-butyl methyl ether; n-Bu n-butyl;n-Bu₃P Tri-n-butylphosphine; NaHMDS Sodium bis(trimethylsilyl)amide; NBSN-Bromosuccinimide; NMP N-Methyl pyrrolidinone; ODCB OrthoDichlorobenzene, or 1,2-dichlorobenzene; Pd(PPh₃)₄ Palladiumtetrakis(triphenylphosphine); Pd₂(dba) ₂Tris(dibenzylideneacetone)dipalladium (0) Ph phenyl; PMSFα-Toluenesulfonyl fluoride; Py or pyr Pyridine; PYBOPBenzotriazol-1-yloxytripyrrolidinophosphonium (or PyBOP)hexafluorophosphate; RPLC Reverse Phase Liquid Chromatography; rt (orRT) Room Temperature; t-Bu tert-Butyl; TBAF Tetrabutylammonium fluoride;TBSCl tert-Butyldimethylsilyl chloride; TFA Trifluoroacetic acid; THFTetrahydrofuran; TIPS Triisopropylsilyl; TMEDAN,N,N′,N′-Tetramethylethylenediamine; TMS Tetramethylsilane; Tr Trityl;and TsOH P-Toluenesulfonic acid.

[0064] These and other aspects of the invention will be apparent fromthe teachings contained herein.

Utility

[0065] The salt forms of the instant invention are useful aspharmaceutical agents for mammals, especially for humans, in thetreatment of tyrosine kinase dependent diseases. Such diseases includethe proliferation of tumor cells, the pathologic neovascularization (orangiogenesis) that supports solid tumor growth, ocularneovascularization (diabetic retinopathy, age-related maculardegeneration, retinal ischemia, macular edema, and the like) andinflammation (psoriasis, rheumatoid arthritis, and the-like). Based onpharmacokinetic studies in animals, the presently claimed salt formshave an unexpectedly superior oral activity profile compared to thecorresponding free base and are therefore particularly suited for oraladministration. They may, however, be adminsitered via other routes asdescribed herein.

[0066] The salt forms of the instant invention may be administered topatients for use in the treatment of cancer. The instant salts inhibittumor angiogenesis, thereby affecting the growth of tumors (J. Rak etal. Cancer Research, 55:4575-4580, 1995). The anti-angiogenesisproperties of the instant salts are also useful in the treatment ofcertain forms of blindness related to retinal vascularization.

[0067] The salt forms of the instant invention are also useful in thetreatment of certain bone-related pathologies, such as osteosarcoma,osteoarthritis, and rickets, also known as oncogenic osteomalacia.(Hasegawa et al., Skeletal Radiol., 28, pp.41-45, 1999; Gerber et al.,Nature Medicine, Vol. 5, No. 6, pp.623-628, June 1999). And since VEGFdirectly promotes osteoclastic bone resorption through KDR/Flk-1expressed in mature osteoclasts (FEBS Let. 473:161-164 (2000);Endocrinology, 141:1667 (2000)), the instant salts are also useful totreat and prevent conditions related to bone resorption, such asosteoporosis and Paget's disease.

[0068] The claimed salt forms can also be used to reduce or preventtissue damage which occurs after cerebral ischemic events, such asstroke, by reducing cerebral edema, tissue damage, and reperfusioninjury following ischemia. (Drug News Perspect 11:265-270 (1998); J.Clin. Invest. 104:1613-1620 (1999).) The salt forms of the instantinvention may also be co-administered with other well known therapeuticagents that are selected for their particular usefulness against thecondition that is being treated. For example, in the case ofbone-related disorders, combinations that would be useful include thosewith antiresorptive bisphosphonates, such as alendronate andrisedronate; integrin blockers (defined further below), such as α_(v)β₃antagonists; conjugated estrogens used in hormone replacement therapy,such as PREMPRO®, PREMARIN® and ENDOMETRION®; selective estrogenreceptor modulators (SERMs), such as raloxifene, droloxifene, CP-336,156(Pfizer) and lasofoxifene; cathespin K inhibitors; and ATP proton pumpinhibitors.

[0069] The instant salt forms are also useful in combination with knownanti-cancer agents. Such known anti-cancer agents include the following:estrogen receptor modulators, androgen receptor modulators, retinoidreceptor modulators, cytotoxic agents, antiproliferative agents,prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIVprotease inhibitors, reverse transcriptase inhibitors, and otherangiogenesis inhibitors.

[0070] “Estrogen receptor modulators” refers to compounds whichinterfere or inhibit the binding of estrogen to the receptor, regardlessof mechanism. Examples of estrogen receptor modulators include, but arenot limited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenylydrazone, and SH646.

[0071] “Androgen receptor modulators” refers to compounds whichinterfere or inhibit the binding of androgens to the receptor,regardless of mechanism. Examples of androgen receptor modulatorsinclude finasteride and other 5α-reductase inhibitors, nilutamide,flutamide, bicalutamide, liarozole, and abiraterone acetate.

[0072] “Retinoid receptor modulators” refers to compounds whichinterfere or inhibit the binding of retinoids to the receptor,regardless of mechanism. Examples of such retinoid receptor modulatorsinclude bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoicacid, a-difluoromethylornithine, ILX23-7553,trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

[0073] “Cytotoxic agents” refer to compounds which cause cell deathprimarily by interfering directly with the cell's functioning or inhibitor interfere with cell myosis, including alkylating agents, tumornecrosis factors, intercalators, microtubulin inhibitors, andtopoisomerase inhibitors.

[0074] Examples of cytotoxic agents include, but are not limited to,tirapazimine, sertenef, cachectin, ifosfamide, tasonermin, lonidamine,carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine,fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin,estramustine, improsulfan tosilate, trofosfamide, nimustine,dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin,cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2-methyl-yridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum (II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin,amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycaminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin.

[0075] Examples of microtubulin inhibitors include paclitaxel, vindesinesulfate, 3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol,rhizoxin; dolastatin, mivobulin isethionate, auristatin, cemadotin,RPR109881, BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) enzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, and BMS188797.

[0076] Some examples of topoisomerase inhibitors are topotecan,hycaptamine, irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,.N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine, (5a, SaB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-155,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-20-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one, anddimesna.

[0077] “Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl) urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-flurouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester; swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N-4-palmitoyl-1-B-D-arabino furanosyl cytosine, and3-aminopyridine-2-carboxaldehyde thiosemicarbazone. “Antiproliferativeagents” also includes monoclonal antibodies to growth factors, otherthan those listed under “angiogenesis inhibitors”, such as trastuzumab,and tumor suppressor genes, such as p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example).

[0078] “HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Compounds which haveinhibitory activity for HMG-CoA reductase can be readily identified byusing assays well-known in the art. For example, see the assaysdescribed or cited in U.S. Pat. No. 4,231,938 at col. 6, and WO 84/02131at pp. 30-33. The terms “HMG-CoA reductase inhibitor” and “inhibitor ofHMG-CoA reductase” have the same meaning when used herein.

[0079] Examples of HMG-CoA reductase inhibitors that may be used includebut are not limited to lovastatin (MEVACOR®; see U.S. Pat. Nos.4,231,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Pat.Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; seeU.S. Pat. Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and5,180,589), fluvastatin (LESCOL®; see U.S. Pat. Nos. 5,354,772,4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896),atorvastatin (LIPITOR®; see U.S. Pat. Nos. 5,273,995, 4,681,893,5,489,691 and 5,342,952) and cerivastatin (also known as rivastatin andBAYCHOL®; see U.S. Pat. No. 5,177,080). The structural formulas of theseand additional HMG-CoA reductase inhibitors that may be used in theinstant methods are described at page 87 of M. Yalpani, “CholesterolLowering Drugs”, Chemistry & Industry, pp. 85-89 (Feb. 5, 1996) and U.S.Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitoras used herein includes all pharmaceutically acceptable lactone andopen-acid forms (i.e., where the lactone ring is opened to form the freeacid) as well as salt and ester forms of compounds which have HMG-CoAreductase inhibitory activity, and therefor the use of such salts,esters, open-acid and lactone forms is included within the scope of thisinvention. An illustration of the lactone portion and its correspondingopen-acid form-is shown below as structures I and II.

[0080] In HMG-CoA reductase inhibitors where an open-acid form canexist, salt and ester forms may preferably be formed from the open-acid,and all such forms are included within the meaning of the term “HMG-CoAreductase inhibitor” as used herein. Preferably, the HMG-CoA reductaseinhibitor is selected from lovastatin and simvastatin, and mostpreferably simvastatin. Herein, the term “pharmaceutically acceptablesalts” with respect to the HMG-CoA reductase inhibitor shall meannon-toxic salts of the compounds employed in this invention which aregenerally prepared by reacting the free acid with a suitable organic orinorganic base, particularly those formed from cations such as sodium,potassium, aluminum, calcium, lithium, magnesium, zinc andtetramethylammonium, as well as those salts formed from amines such asammonia, ethylenediamine, N-methylglucamine, lysine, arginine,ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine,diethanolamine; procaine, N-benzylphenethylamine,1-p-chlorobenzyl-2-pyrrolidine-1′-yl-methylbenzimidazole, diethylamine,piperazine, and tris(hydroxymethyl) aminomethane. Further examples ofsalt forms of HMG-CoA reductase inhibitors may include, but are notlimited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, calcium edetate, camsylate, carbonate,chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynapthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote,palmitate, panthothenate, phosphate/diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, and valerate.

[0081] Ester derivatives of the described HMG-CoA reductase inhibitorcompounds may act as prodrugs which, when absorbed into the bloodstreamof a warm-blooded animal, may cleave in such a manner as to release thedrug form and permit the drug to afford improved therapeutic efficacy.

[0082] “Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase). Examples of prenyl-protein transferase inhibiting compoundsinclude(+)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(−)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(+)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,5(S)-n-butyl-1-(2,3-dimethylphenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,(S)-1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-5-[2-(ethanesulfonyl)methyl)-2-piperazinone,5(S)-n-Butyl-1-(2-methylphenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-2-methyl-5-imidazolylmethyl]-2-piperazinone,1-(2,2-diphenylethyl)-3-[N-(1-(4-cyanobenzyl)-1H-imidazol-5-ylethyl)carbamoyl]piperidine,4-{5-[4-Hydroxymethyl-4-(4-chloropyridin-2-ylmethyl)-piperidine-1-ylmethyl]-2-methylimidazol-1-ylmethylbenzonitrile,4-{5-[4-hydroxymethyl-4-(3-chlorobenzyl)-piperidine-1-ylmethyl]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-pyridin-1-yl)benzyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-{3-[4-(5-chloro-2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-{3-[4-(2-Oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-[3-(2-Oxo-1-phenyl-1,2-dihydropyridin-4-ylmethyl)-3H-imidazol-4-ylmethyl}benzonitrile,18,19-dihydro-19-oxo-5H,17H-6,10:12,16-dimetheno-1H-imidazo[4,3-c][1,11,4]dioxaazacyclo-nonadecine-9-carbonitrile,(±)-19,20-Dihydro-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriaza-cyclooctadecine-9-carbonitrile,19,20-dihydro-19-oxo-5H,17H-18,21-ethano-6,10:12,16-dimetheno-22H-imidazo[3,4-h][1,8,11,14]oxatriazacycloeicosine-9-carbonitrile,and(±)-19,20-Dihydro-3-methyl-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxa-triazacyclooctadecine-9-carbonitrile.

[0083] Other examples of prenyl-protein transferase inhibitors can befound in the following publications and patents: WO 96/30343, WO97/18813, WO 97/21701, WO 97/23478, WO97/38665, WO-98/28980, WO98/29119, WO 95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430,U.S. Pat. No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No.5,589,485, U.S. Pat. No. 5,602,098, European Patent Publ. 0 618 221,European Patent Publ. 0 675 112, European Patent Publ. 0 604 181,European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO 95/11917,WO 95/12612, WO 95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO96/05169, WO 96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO97/44350, WO 98/02436, and U.S. Pat. No. 5,532,359. For an example ofthe role of a prenyl-protein transferase inhibitor on angiogenesis seeEuropean J. of Cancer, Vol. 35, No. 9, pp.1394-1401 (1999).

[0084] Examples of HIV protease inhibitors include amprenavir, abacavir,CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, tipranavir,ritonavir, saquinavir, ABT-378, AG 1776, and BMS-232,632. Examples ofreverse transcriptase inhibitors include delaviridine, efavirenz,GS-840, HB Y097, lamivudine, nevirapine, AZT, 3TC, ddC, and ddI.

[0085] “Angiogenesis inhibitors” refers to compounds that inhibit theformation of new blood vessels, regardless of mechanism. Examples ofangiogenesis inhibitors include, but are not limited to, tyrosine kinaseinhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1(VEGFR1) and Flk-1/KDR (VEGFR20), inhibitors of epidermal-derived,fibroblast-derived, or platelet derived growth factors, MMP (matrixmetalloprotease) inhibitors, integrin blockers, interferon-α,interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors,including nonsteroidal anti-inflammatories (NSAIDs) like aspirin andibuprofen as well as selective cyclooxygenase-2 inhibitors likecelecoxib-and rofecoxib (PNAS, Vol. 89, p. 7384. (1992); JNCI, Vol. 69,p. 475 (1982); Arch. Opthalmol., Vol. 108, p.573 (1990); Anat. Rec.,Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin,Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p.107(1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol.57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med.,Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)),carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab.Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, NatureBiotechnology, Vol. 17, pp.963-968 (October 1999); Kim et al., Nature,362, 841-844 (1993) WO 00/44777; and WO 00/61186).

[0086] Other therapeutic agents that modulate or inhibit angiogenesisand may also be used in combination with the salt forms of the instantinvention include agents that modulate or inhibit the coagulation andfibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692(2000)). Examples of such agents that modulate or inhibit thecoagulation and fibrinolysis pathways include, but are not limited to,heparin (see Thromb. Haemost. 80:10-23 (1998)), low molecular weightheparins and carboxypeptidase U inhibitors (also known as inhibitors ofactive thrombin activatable fibrinolysis inhibitor [TAFIa]) (seeThrombosis Res. 101:329-354 (2001)). TAFIa inhibitors have beendescribed in U.S. Ser. Nos. 60/310,927 (filed Aug. 8, 2001) and60/349,925(filed Jan. 18, 2002).

[0087] As described above, the combinations with NSAID's are directed tothe use of NSAID's which are potent COX-2 inhibiting agents. Forpurposes of this specification an NSAID is potent if it possess an IC50for the inhibition of COX-2 of 1 μM or less as measured by the cell ormicrosomal assay disclosed herein.

[0088] The invention also encompasses combinations with NSAID's whichare selective COX-2 inhibitors. For purposes of this specificationNSAID's which are selective inhibitors of COX-2 are defined as thosewhich possess a specificity for inhibiting COX-2 over COX-1 of at least100 fold as measured by the ratio of IC₅₀ for COX-2 over IC₅₀ for COX-1evaluated by the cell or microsomal assay disclosed hereinunder. Suchcompounds include, but are not limited to those disclosed in U.S. Pat.No. 5,474,995, issued Dec. 12, 1995, U.S. Pat. No. 5,861,419, issuedJan. 19, 1999, U.S. Pat. No. 6,001,843, issued Dec. 14, 1999, U.S. Pat.No. 6,020,343, issued Feb. 1, 2000, U.S. Pat. No. 5,409,944, issued-Apr.25, 1995, U.S. Pat. No. 5,436,265, issued Jul. 25, 1995, U.S. Pat. No.5,536,752, issued Jul. 16, 1996, U.S. Pat. No. 5,550,142, issued Aug.27, 1996, U.S. Pat. No. 5,604,260, issued Feb. 18, 1997, U.S. Pat. No.5,698,584, issued Dec. 16, 1997, U.S. Pat. No. 5,710,140, issued Jan.20, 1998, WO 94/15932, published Jul. 21, 1994, U.S. Pat. No. 5,344,991,issued Jun. 6, 1994, U.S. Pat. No. 5,134,142, issued Jul. 28, 1992, U.S.Pat. No. 5,380,738, issued Jan. 10, 1995, U.S. Pat. No. 5,393,790,issued Feb. 20, 1995, U.S. Pat. No. 5,466,823, issued Nov. 14, 1995(Celebrex), U.S. Pat. No. 5,633,272, issued May 27, 1997 (Valdecoxib),and U.S. Pat. No. 5,932,598, issued Aug. 3, 1999 (Parecoxib), all ofwhich are hereby incorporated by reference.

[0089] Inhibitors of COX-2 that are particularly useful in the instantmethod of treatment are:

[0090] 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and

[0091]5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine;

[0092] or a pharmaceutically acceptable salt thereof.

[0093] General and specific synthetic procedures for the preparation ofthe COX-2 inhibitor compounds described above are found in U.S. Pat. No.5,474,995, issued Dec. 12, 1995, U.S. Pat. No. 5,861,419, issued Jan.19, 1999, and U.S. Pat. No. 6,001,843, issued Dec. 14, 1999, all ofwhich are herein incorporated by reference.

[0094] Compounds that have been described as specific inhibitors ofCOX-2 and are therefore useful in the present invention include, but arenot limited to, the following:

[0095] or a pharmaceutically acceptable salt thereof.

[0096] Compounds, which are described as specific inhibitors of COX-2and are therefore useful in the present invention, and methods ofsynthesis thereof, can be found in the following patents, pendingapplications and publications, which are herein incorporated byreference: WO 94/15932, published Jul. 21, 1994, U.S. Pat. No.5,344,991, issued Jun. 6, 1994, U.S. Pat. No. 5,134,142, issued Jul. 28,1992, U.S. Pat. No. 5,380,738, issued Jan. 10, 1995, U.S. Pat. No.5,393,790, issued Feb. 20, 1995, U.S. Pat. No. 5,466,823, issued Nov.14, 1995 (Celebrex), U.S. Pat. No. 5,633,272, issued May 27, 1997(Valdecoxib), and U.S. Pat. No. 5,932,598, issued Aug. 3, 1999(Parecoxib).

[0097] Compounds which are specific inhibitors of COX-2 and aretherefore useful in the present invention, and methods of synthesisthereof, can be found in the following patents, pending applications andpublications, which are herein incorporated by reference: U.S. Pat. No.5,474,995 issued Dec. 12, 1995, U.S. Pat. No. 5,861,419 issued Jan. 19,1999, U.S. Pat. No. 6,001,843 issued Dec. 14, 1999, U.S. Pat. No.6,020,343 issued Feb. 1, 2000, U.S. Pat. No. 5,409,944 issued Apr. 25,1995, U.S. Pat. No. 5,436,265 issued Jul. 25, 1995, U.S. Pat. No.5,536,752 issued Jul. 16, 1996, U.S. Pat. No. 5,550,142 issued Aug. 27,1996, U.S. Pat. No. 5,604,260 issued Feb. 18, 1997, U.S. Pat. No.5,698,584 issued Dec. 16, 1997, and U.S. Pat. No. 5,710,140 issued Jan.20, 1998.

[0098] Other examples of angiogenesis inhibitors include, but are notlimited to, endostation, ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RP14610, NX31838, sulfatedmannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

[0099] As used above, “integrin blockers” refers to compounds whichselectively antagonize, inhibit or counteract binding of a physiologicalligand to the α_(v)β₃ integrin, to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe αvβ5 integrin, to compounds which antagonize, inhibit or counteractbinding of a physiological ligand to both the αvβ3 integrin and the αvβ5integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the αvβ6,αvβ8, α1β1, α2β1, α5β1, α₆β1 and α6β4 integrins. The term also refers toantagonists of any combination of αvβ3, αvβ5, αvβ6, αvβ8, α1β1, α2β1,α5β1, α6β1 and α6β4 integrins.

[0100] Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, STI571, CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, ST1571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

[0101] The instantly claimed salt forms are also useful, alone or incombination with platelet fibrinogen receptor (GP IIb/IIIIa)antagonists, such as tirofiban, to inhibit metastasis of cancerouscells. Tumor cells can activate platelets largely via thrombingeneration. This activation is associated with the release of VEGF. Therelease of VEGF enhances metastasis by increasing extravasation atpoints of adhesion to vascular endothelium (Amirkhosravi, Platelets 10,285-292, 1999). Therefore, the present salts can serve to inhibitmetastasis, alone or in combination with GP IIb/IIIa) antagonists.Examples of other fibrinogen receptor antagonists include abciximab,eptifibatide, sibrafiban, lamifiban, lotrafiban, cromofiban, andCT50352.

Formulation

[0102] The salt forms of the instant invention may be administered tomammals, preferably humans, either alone or, preferably, in combinationwith pharmaceutically acceptable carriers or diluents, optionally withknown adjuvants, such as alum, in a pharmaceutical composition,according to standard pharmaceutical practice. The salt forms can beadministered orally or parenterally, including the intravenous,intramuscular, intraperitoneal, subcutaneous, rectal and topical routesof administration.

[0103] For oral use of a chemotherapeutic compound according to thisinvention, the selected salt may be administered, for example, in theform of tablets or capsules, or as an aqueous solution or suspension. Inthe case of tablets for oral use, carriers-which are commonly usedinclude lactose and cornstarch, and lubricating agents, such asmagnesium stearate, are commonly added. For oral administration incapsule form, useful diluents include lactose and dried cornstarch. Whenaqueous suspensions are required for oral use, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring agents may be added. For intramuscular,intraperitoneal, subcutaneous and intravenous use, sterile solutions ofthe active ingredient are usually prepared, and the pH of the solutionsshould be suitably adjusted and buffered. For intravenous use, the totalconcentration of solutes should be controlled in order to render thepreparation isotonic.

[0104] If formulated as a fixed dose, combination products, such asthose described hereinabove, employ the salts of this invention withinthe dosage range described below and the other pharmaceutically activeagent(s) within its approved dosage range. Salt forms of the instantinvention may alternatively be used sequentially with knownpharmaceutically acceptable agent(s) when a combination formulation isinappropriate.

[0105] The term “administration” and variants thereof (e.g.,“administering” a compound) in reference to a salt of the inventionmeans introducing the salt or a prodrug of the salt into the system ofthe animal in need of treatment. When a compound of the invention orprodrug thereof is provided in combination with one or more other activeagents (e.g., a cytotoxic agent, etc.), “administration” and itsvariants are each understood to include concurrent and sequentialintroduction of the salt or prodrug thereof and other agents.

[0106] As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

[0107] The term “therapeutically effective amount” as used herein meansthat amount of active salt, or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician.

[0108] The present invention also encompasses a pharmaceuticalcomposition useful in the treatment of cancer, comprising theadministration of a therapeutically effective amount of the salt formsof this invention, with or without pharmaceutically acceptable carriersor diluents. Suitable compositions of this invention include aqueoussolutions comprising-salts of this invention andpharmacologically-acceptable carriers, e.g., saline, at a pH level,e.g., 7.4.

[0109] When a salt according to this invention is administered into ahuman subject, the daily dosage will normally be determined by theprescribing physician with the dosage generally varying according to theage, weight, and response of the individual patient, as well as theseverity of the patient's symptoms.

[0110] In one exemplary application, a suitable amount of a salt isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount between about 0.1 mg/kg of body weight to about 60mg/kg of body weight per day, preferably of between 0.5 mg/kg of bodyweight to about 40 mg/kg of body weight per day.

Assays

[0111] The salt forms of the instant invention described in the Exampleswere tested by the assays described below and were found to have kinaseinhibitory activity. Other assays are known in the literature and couldbe readily performed by those of skill in the art (see, for example,Dhanabal et al., Cancer Res. 59:189-197; Xin et al., J. Biol. Chem.274:9116-9121; Sheu et al., Anticancer Res. 18:4435-4441; Ausprunk etal., Dev. Biol. 38:237-248; Gimbrone et al., J. Natl. Cancer Inst.52:413-427; Nicosia et al., In Vitro 18:538-549).

[0112] I. VEGF Receptor Kinase Assay

[0113] VEGF receptor kinase activity is measured by incorporation ofradio-labeled phosphate into polyglutamic acid, tyrosine, 4:1 (pEY)substrate. The phosphorylated pEY product is trapped onto a filtermembrane and the incorporation of radio-labeled phosphate quantified byscintillation counting.

[0114] Materials

[0115] VEGF Receptor Kinase

[0116] The intracellular tyrosine kinase domains of human KDR (Terman,B. I. et al. Oncogene (1991) vol. 6, pp. 1677-1683.) and Flt-1 (Shibuya,M. et al. Oncogene (1990) vol. 5, pp. 519-524) were cloned asglutathione S-transferase (GST) gene fusion proteins. This wasaccomplished by cloning the cytoplasmic domain of the KDR kinase as anin frame fusion at the carboxy terminus of the GST gene. Solublerecombinant GST-kinase domain fusion proteins were expressed inSpodoptera frugiperda (Sf21) insect cells (Invitrogen) using abaculovirus expression vector (pAcG2T, Pharmingen).

[0117] The other materials used and their compositions were as follows:

[0118] Lysis buffer: 50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM EDTA,0.5% triton X-100, 10% glycerol, 10 mg/mL of each leupeptin, pepstatinand aprotinin and 1 mM phenylmethylsulfonyl fluoride (all Sigma).

[0119] Wash buffer: 50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM EDTA,0.05% triton X-100, 10% glycerol, 10 mg/mL of each leupeptin, pepstatinand aprotinin and 1 mM phenylmethylsulfonyl fluoride.

[0120] Dialysis buffer: 50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mMEDTA, 0.05% triton X-100, 50% glycerol, 10 mg/mL of each leupeptin,pepstatin and aprotinin and 1 mM phenylmethylsuflonyl fluoride.

[0121] 10× reaction buffer: 200 mM Tris, pH 7.4, 1.0 M NaCl, 50 mMMnCl2, 10 mM DTT and 5 mg/mL bovine serum albumin (Sigma).

[0122] Enzyme dilution buffer: 50 mM Tris, pH 7.4, 0.1 M NaCl, 1 mM DTT,10% glycerol, 100 mg/mL BSA.

[0123] 10× Substrate: 750 μg/mL poly (glutamic acid, tyrosine; 4:1)(Sigma).

[0124] Stop solution: 30% trichloroacetic acid, 0.2 M sodiumpyrophosphate (both Fisher).

[0125] Wash solution: 15% trichloroacetic acid, 0.2 M sodiumpyrophosphate.

[0126] Filter plates: Millipore #MAFC NOB, GF/C glass fiber 96 wellplate.

[0127] Method

[0128] A. Protein Purification

[0129] 1. Sf21 cells were infected with recombinant virus at amultiplicity of infection of 5 virus particles/cell and grown at 27° C.for 48 hours.

[0130] 2. All steps were performed at 4° C. Infected cells wereharvested by centrifugation at 1000×g and lysed at 4° C. for 30 minuteswith {fraction (1/10)} volume of lysis buffer followed by centrifugationat 100,000×g for 1 hour. The supernatant was then passed over aglutathione Sepharose-column-(Pharmacia) equilibrated in lysis bufferand washed with 5 volumes of the same buffer followed by 5 volumes ofwash buffer. Recombinant GST-KDR protein was eluted with wash buffer/10mM reduced glutathione (Sigma) and dialyzed against dialysis buffer.

[0131] B. VEGF Receptor Kinase Assay

[0132] 1) Add 5 μl of inhibitor or control to the assay in 50% DMSO.

[0133] 2) Add 35 μl of reaction mix containing 5 μl of 10× reactionbuffer, 5 μl 25 mM ATP/10 μCi [³³P]ATP (Amersham), and 5 μl 10×substrate.

[0134] 3) Start the reaction by the addition of 10 μl of KDR (25 nM) inenzyme dilution buffer.

[0135] 4) Mix and incubate at room temperature for 15 minutes.

[0136] 5) Stop by the addition of 50 μl stop solution.

[0137] 6) Incubate for 15 minutes at 4° C.

[0138] 7) Transfer a 90 μl aliquot to filter plate.

[0139] 8) Aspirate and wash 3 times with wash solution.

[0140] 9) Add 30 μl of scintillation cocktail, seal plate and count in aWallac Microbeta scintillation counter.

[0141] II. Human Umbilical Vein Endothelial Cell Mitogenesis Assay

[0142] Human umbilical vein endothelial cells (HUVECs) in cultureproliferate in response to VEGF treatment and can be used as an assaysystem to quantify the effects of KDR kinase inhibitors on VEGFstimulation. In the assay described, quiescent HUVEC monolayers aretreated with vehicle or test compound 2 hours prior to addition of VEGFor basic fibroblast growth factor (bFGF). The mitogenic response to VEGFor bFGF is determined by measuring the incorporation of [³H] thymidineinto cellular DNA.

[0143] Materials

[0144] HUVECs: HUVECs frozen as primary culture isolates are obtainedfrom Clonetics Corp. Cells are maintained in Endothelial Growth Medium(EGM; Clonetics) and are used for mitogenic assays described in passages3-7 below.

[0145] Culture Plates: NUNCLON 96-well polystyrene tissue culture plates(NUNC #167008).

[0146] Assay Medium: Dulbecco's modification of Eagle's mediumcontaining 1 g/mL glucose (low-glucose DMEM; Mediatech) plus 10% (v/v)fetal bovine serum (Clonetics).

[0147] Test Compounds: Working stocks of test compounds are dilutedserially in 100% dimethylsulfoxide (DMSO) to 400-fold greater than theirdesired final concentrations. Final dilutions to 1× concentration aremade directly into Assay Medium immediately prior to addition to cells.

[0148] 10× Growth Factors: Solutions of human VEGF₁₆₅ (500 ng/mL; R&DSystems) and bFGF (10 ng/mL; R&D Systems) are prepared in Assay Medium.

[0149] 10× [³H]Thymidine: [Methyl-³H]thymidine (20 Ci/mmol; Dupont-NEN)is diluted to 80 μCi/mL in low-glucose DMEM.

[0150] Cell Wash Medium: Hank's balanced salt solution (Mediatech)containing 1 mg/mL bovine serum albumin (Boehringer-Mannheim).

[0151] Cell Lysis Solution: 1 N NaOH, 2% (w/v) Na₂CO₃.

[0152] Method

[0153] 1. HUVEC monolayers maintained in EGM are harvested bytrypsinization and plated at a density of 4000 cells per 100 μL AssayMedium per well in 96-well plates. Cells are growth-arrested for 24hours at 37° C. in a humidified atmosphere containing 5% CO₂.

[0154] 2. Growth-arrest medium is replaced by 100 μL Assay Mediumcontaining either vehicle (0.25% [v/v] DMSO) or the desired finalconcentration of test compound. All determinations are performed intriplicate. Cells are then incubated at 37° C. with 5% CO₂ for 2 hoursto allow test compounds to enter cells.

[0155] 3. After the 2-hour pretreatment period, cells are stimulated byaddition of 10 μL/well of either Assay Medium, 10×VEGF solution or10×bFGF solution. Cells are then incubated at 37° C. and 5% CO₂.

[0156] 4. After 24 hours in the presence of growth factors, 10×[³H]thymidine (10 mL/well) is added.

[0157] 5. Three days after addition of [³H]thymidine, medium is removedby aspiration, and cells are washed twice with Cell Wash Medium (400μL/well followed by 200 μL/well). The washed, adherent cells are thensolubilized by addition of Cell Lysis Solution (100 μL/well) and warmingto 37° C. for 30 minutes. Cell lysates are transferred to 7-mL glassscintillation vials containing 150 μL of water. Scintillation cocktail(5 mL/vial) is added, and cell-associated radioactivity is determined byliquid scintillation spectroscopy.

[0158] Based upon the foregoing assays the salt forms of the instantinvention are inhibitors of VEGF and thus are useful for the inhibitionof angiogenesis, such as in the treatment of ocular disease, e.g.,diabetic retinopathy and in the treatment of cancers, e.g., solidtumors. The instant salt forms inhibit VEGF-stimulated mitogenesis ofhuman vascular endothelial cells in culture with IC₅₀ values between0.01-5.0 μM. These salt forms may also show selectivity over relatedtyrosine kinases (e.g., FGFR1 and the Src family; for relationshipbetween Src kinases and VEGFR kinases, see Eliceiri et al., MolecularCell, Vol. 4, pp.915-924, December 1999).

[0159] III. Flt-1 Kinase Assay

[0160] Flt-1 was expressed as a GST fusion to the Flt-i kinase domainand was expressed in baculovirus/insect cells. The following protocolwas employed to assay compounds for Flt-1 kinase inhibitory activity:

[0161] 1) Inhibitors were diluted to account for the final dilution inthe assay, 1:20.

[0162] 2) The appropriate amount of reaction mix was prepared at roomtemperature:

[0163] 10× Buffer (20 mM Tris pH 7.4/0.1 M NaCl/1 mM DTT final)

[0164] 0.1M MnCl₂ (5 mM final)

[0165] pEY substrate (75 μg/mL)

[0166] ATP/[³³P]ATP (2.5 μM/1 μCi final)

[0167] BSA (500 μg/mL final).

[0168] 3) 5 μL of the diluted inhibitor was added to the reaction mix.(Final volume of 5 μL in 50% DMSO). To the positive control wells, blankDMSO (50%) was added.

[0169] 4) 35 μL of the reaction mix was added to each well of a 96 wellplate.

[0170] 5) Enzyme was diluted into enzyme dilution buffer (kept at 4°C.).

[0171] 6) 10 μL of the diluted enzyme was added to each well and mix (5nM final). To the negative control wells, 10 μL 0.5 M EDTA was added perwell instead (final 100 mM).

[0172] 7) Incubation was then carried out at room temperature for 30minutes.

[0173] 8) Stopped by the addition of an equal volume (50 μL) of 30%TCA/0.1 M Na pyrophosphate.

[0174] 9) Incubation was then carried out for 15 minutes to allowprecipitation.

[0175] 10) Transfered to Millipore filter plate.

[0176] 11) Washed 3×with 15% TCA/0.1M Na pyrophosphate (125 μL perwash).

[0177] 12) Allowed to dry under vacuum for 2-3 minutes.

[0178] 13) Dryed in hood for about 20 minutes.

[0179] 14) Assembled Wallac Millipore adapter and added 50 μL ofscintillant to each well and counted.

[0180] IV. Flt-3 Kinase Assay

[0181] Flt-3 was expressed as a GST fusion to the Flt-3 kinase domain,and was expressed in baculovirus/insect cells. The following protocolwas employed to assay compounds for Flt-3 kinase inhibitory activity:

[0182] 1) Dilute inhibitors (account for the final dilution into theassay, 1:20)

[0183] 2) Prepare the appropriate amount of reaction mix at roomtemperature.

[0184] 10× Buffer (20 mM Tris pH 7.4/0.1 M NaCl/1 mM DTT final)

[0185] 0.1M MnCl2 (5 mM final)

[0186] pEY substrate (75 μg/mL)

[0187] ATP/[³³P]ATP (0.5 μM/L μCi final)

[0188] BSA (500 μg/mL final)

[0189] 3) Add 5 μL of the diluted inhibitor to the reaction mix. (Finalvolume of 5 μL in 50% DMSO). Positive control wells add blank DMSO(50%).

[0190] 4) Add 35 μL of the reaction mix to each well of a 96 well plate.

[0191] 5) Dilute enzyme into enzyme dilution buffer (keep at 4° C.).

[0192] 6) Add 10 μL of the diluted enzyme to each well and mix (5-10 nMfinal). Negative control wells—add 10 μL 0.5 M EDTA per well instead(final 100 mM)

[0193] 7) Incubate at room temperature for 60 minutes.

[0194] 8) Stop by the addition of an equal volume (50 μL) of 30%TCA/0.1M Na pyrophosphate.

[0195] 9) Incubate for 15 minutes to allow precipitation.

[0196] 10) Transfer to Millipore filter plate.

[0197] 11) Wash 3× with 15% TCA/0.1M Na pyrophosphate (125 μL per wash).

[0198] 12) Allow to dry under vacuum for 2-3 minutes.

[0199] 13) Dry in hood for about 20 minutes.

[0200] 14) Assemble Wallac Millipore adapter and add 50 μL ofscintillant to each well and count.

EXAMPLES

[0201] Examples provided are intended to assist in a furtherunderstanding of the invention. Particular materials employed, speciesand conditions are intended to be further illustrative of the inventionand not limiting of the reasonable scope thereof.

[0202] The free bases used to prepare the salt forms of this inventionmay be obtained by employing the procedures described below, as well asthose described in WO 01/17995, published Mar. 15, 2001, herebyincorporated by reference. In addition, other procedures may be usedsuch as standard manipulations of reactions that are known in theliterature.

[0203] HPLC Methods Used: HPLC Analysis: Isocratic method (forsolubility studies) Analysis Method Chromatographic Conditions (HPLC):Column: BDS HYPESIL, C18 (250 mm × 46 mm), 5 μm particle size Columnambient Temperature: Detector: 230 nm (UV wavelength) Column Temp.Ambient Flow Rate: 1.0 mL/min Injection Volume: 20 μL Mobile Phase: A)0.1% Phosphoric Acid B) 100% Acetonitrile Diluent: 50% Acetonitrile-DIwater Gradient Profile: (A/B) starts from (60/40) and stays at (60/40)for 10 minutes. Run Time: 10 minutes

[0204]

[0205] 2-Chloro-thiazole-5-carbonitrile (1-2)

[0206] A flame dried round bottom flask under N₂ was charged with 150 mLanhydrous MeCN. CuCl₂ (12.9 g, 95.9 mmol, 1.2 equiv) was added and thereaction was maintained in a room temperature bath tert-Butylnitrite(14.3 mL, 120 mmol, 1.5 equiv) was added gradually over 10 minutes.After 10 minutes, 2-amino-thiazole-5-carbonitrile (1-1, 10.0 g, 79.9mmol) was added as a solid gradually. The reaction was stirred at roomtemperature for 4 hours. The reaction was poured into 400 mL 0.5M HCl(aq). The mixture was extracted 3× with EtOAc. The organic phases weredried over Na₂SO₄, filtered and concentrated to afford pure desiredproduct.

[0207]¹H NMR (CDCl₃) δ 8.04 (s).

[0208] 2-Acetylamino-isonicotinic Acid (2-2)

[0209] N-(4-Methyl-pyridin-2-yl)-acetamide, 70 g (466 mmol) was stirredin 400 mL water. The mixture was warmed to 80° C. KMnO₄ (368 g, 2.33mol, 5 equiv) was added dissolved in water over 45 minutes. The solutionwas heated to reflux for 3 hours. The reaction was then cooled andfiltered. The filtrate was concentrated in vacuo to afford the desiredproduct. ¹H NMR (CD₃OD) δ 8.62 (s, 1H), 8.42 (d, 1H, J=5.1 Hz), 7.59(dd, 1H, J=5.1 Hz), 2.19 (s, 3H).

[0210] 2-Amino-isonicotinic Acid Methyl Ester (2-3)

[0211] 2-Acetylamino-isonicotinic acid (3.10 g, 17.2 mmol) was stirredin 35 mL MeOH at 0° C. HCl (g) was bubbled through the solution for 10minutes and then the reaction was heated to reflux. After 16 hours thereaction was concentrated in vacuo. The residue was diluted with waterand the pH was adjusted to 7 with Na₂CO₃ (s). A white precipitate formedwhich was filtered to afford a portion of pure desired product. Theaqueous phase was extracted three times with 95:5 dichloromethane(DCM)/nBuOH. The organic phases were dried over Na₂SO₄, filtered andconcentrated to afford more of the pure product as a white solid.

[0212]¹H NMR (CDCl₃) δ 8.19 (d, 1H, J=5.3 Hz), 7.17 (dd, 1H, J=1.4, 5.3Hz), 7.07 (d, 1H, J=1.3 Hz), 4.64 (bs, 2H), 3.92 (s, 3H). MS[M+H]+=153.0.

[0213] (2-Amino-pyridin-4-yl)-methanol (2-4)

[0214] 2-Amino-isonicotinic acid methyl ester (6.0 g, 39.4 mmol) wasdissolved in 80 mL anhydrous THF in a flame dried round bottom flaskunder nitrogen gas. The solution was cooled to −45° C. and LAH (39.4 mL,1M in THF) was added slowly. The reaction was allowed to warm to 0° C.and was quenched by the addition of 15 mL of 1M NaOH (aq). The solutionwas filtered and the solid was washed with THF. The filtrate wasconcentrated to afford the pure product.

[0215]¹H NMR (DMSO-d₆) δ 7.79 (d, 1H, J=5.2 Hz), 6.41 (s, 1H), 6.38 (d,1H, J=5.9 Hz), 5.79 (bs, 2H), 5.19 (t, 2H, J=5.7), 4.35 (d, 2H, J=5.6Hz).

[0216] 4-(tert-Butyl-dimethyl-silanyloxymethyl)-pyridin-2-ylamine (2-5)

[0217] (2-Amino-pyridin-4-yl)-methanol (4.68 g, 37.7 mmol) was dissolvedin 40 mL anhydrous DMF under N₂. Imidazole (2.57 g, 37.7 mmol, 1 equiv)was added followed by the addition of TBSCI (5.68 g, 37.7 mmol, 1equiv). After 2 hours the reaction was quenched by the addition ofwater. A precipitate formed which was filtered to afford pure desiredproduct. The aqueous filtrate was extract 3× with EtOAc. The organicphases were dried over Na₂SO₄, filtered and concentrated to affordadditional impure material. ¹H NMR (CDCl₃) δ 7.99 (d, 1H, J=5.8 Hz),6.57 (d, 1H, J=5.1 Hz), 6.51 (s, 1H), 4.64 (s, 2H), 4.40 (bs, 2H), 0.95(s, 9H), 0.11 (s, 6H).

[0218]2-[4-(tert-Butyl-dimethyl-silanyloxymethyl)-pyridin-2-ylamino]-thiazole-5-carbonitrile(3-1)

[0219] 4-(tert-Butyl-dimethyl-silanyloxymethyl)-pyridin-2-ylamine (2-5,5.94 g, 24.9 mmol) was dissolved in 50 mL anhydrous tetrahydrofuran(THF) under N₂. NaH (60% suspension, 2.99 g, 74.8 mmol, 3 equiv) wasadded (vigorous bubbling occurred) and the resulting mixture was stirredfor 15 minutes. 2-Chloro-thiazole-5-carbonitrile (1-2, 4.32 g, 29.9mmol) was added and the reaction was heated to reflux. After 2 hours thereaction was cooled and was quenched by the addition of water. The THFwas removed in vacuo and the resulting aqueous solution was adjusted topH=7 by the addition of 1M HCl (aq). The resulting precipitate wasfiltered and washed with water to provide reasonably pure product. ¹HNMR (CDCl₃) δ 10.32 (bs, 1H), 8.33 (d, 1H, J=5.3 Hz), 7.99 (s, 1H), 6.96(s, 1H), 6.91 (d, 1H, J=5.3 Hz), 4.78 (s, 2H), 0.98 (s, 9H), 0.16 (s,6H).

[0220] 2-(4-Hydroxymethyl-pyridin-2-ylamino)-thiazole-5-carbonitrile(3-2)

[0221]2-[4-(tert-Butyl-dimethyl-silanyloxymethyl)-pyridin-2-ylamino]-thiazole-5-carbonitrile(1.30 g, 3.75 mmol) was dissolved in 10 mL anh THF. Hydrogen-fluoride(Aldrich, 5.0 mL) was added and the reaction was stirred for 20 minutes.The bulk of the solvent was removed in vacuo and the resulting residuewas diluted with half-saturated NaHCO₃ (aq). A precipitate formed whichwas filtered and washed with water to afford the titled compound. ¹H NMR(DMSO-d₆) δ 12.23 (bs, 1H), 8.30 (d, 1H, J=5.3 Hz), 8.26 (s, 1H), 7.15(s, 1H), 6.99 (d, 1H, J=5.3 Hz), 5.49 (t, 1H, J=5.7 Hz) 4.54 (d, 2H,J=5.7 Hz).

[0222] 2-(4-Chloromethyl-pyridin-2-ylamino)-thiazole-5-carbonitrile(3-3)

[0223] 2-(4-Hydroxymethyl-pyridin-2-ylamino)-thiazole-5-carbonitrile(0.883 g, 3.80 mmol) was stirred in anhydrous CH₂Cl₂ (12 mL) under N₂.Dimethyl-formamide (0.354 mL, 3.80 mmol, 1 equiv) was added followed bythe addition of phosphorous oxychloride (0.294 mL, 3.80 mmol). After 4hours the reaction was concentrated and quenched by the addition ofsaturated NaHCO₃ (aq). A precipitate formed which was filtered andwashed with water to provide the titled compound. ¹H NMR (DMSO-d₆) δ12.35 (bs, 1H), 8.40 (d, 1H, J=5.3 Hz), 8.28 (s, 1H), 7.20 (s, 1H), 7.12(d, 1H, J=5.3 Hz), 4.82 (s, 2H).

[0224] To a solution of Boc-piperazine, 4-1, in CH₂Cl₂ (200 mL) wasadded 6.74 g (1 equiv) methylisocyanate in CH₂Cl₂ (50 mL). The reactionmixture was stirred at room temperature for 6 hours and another 0.25 eq(1.69 g) of methylisocyanate was added. The reaction mixture was thenstirred at room temperature overnight. The reaction was subsequentlyquenched with water (75 mL) and extracted with CH₂Cl₂ (3×50 mL). Thecombined organics were dried over Na₂SO₄, filtered, and concentrated toafford 4-2 as a white solid. ¹H NMR (CDCl₃) δ 4.44 (bs, 1H), 3.48-3.33(m, 8H), 2.82 (d, 3H, J=4.58), 1.47 (s, 9H).

[0225] To a solution of 4-2 in CH₂Cl₂ at 0° C. was added excess 4.0M HCl(101.5 mL, 406 mmol, 3.5 equiv) in dioxane. The reaction mixture wasallowed to warm to room temperature and was stirred for 4 hours. Themixture was then concentrated to afford1-[(methylamino)carbonyl]piperazin-4-ium chloride, the HCl salt of 4-3,as an off white solid. ¹H NMR (DMSO-d₆) δ 9.28 (bs, 1H), 7.94 (bs, 1H),3.52 (m, 4H), 3.01 (m, 4H), 2.57 (s, 3H).2-(4-Chloromethyl-pyridin-2-ylamino)-thiazole-5-carbonitrile 3-3 (8.00g, 31.9 mmol) was stirred in 60 mL DMSO.1-[(Methylamino)carbonyl]piperazin-4-ium chloride (11.5 g, 63.8 mmol)was added, followed by addition of triethylamine (13.34 mL, 95.7 mmol).The reaction was allowed to stir at room temperature for 15 hours, atwhich time an additional 2.00 g piperazine hydrochloride (11.1 mmol) wasadded. No further progress was observed so the reaction was warmed to45° C. but there was still no further progress. The reaction was cooledto room temperature. An additional 6.6 mL Et₃N (48 mmol) was then added.After an additional hour, the reaction was diluted with 300 mL water.The resulting precipitate was filtered, washed with water and air dried.The solid was purified by flash chromatography (eluted with 92:8DCM/MeOH) to afford the product 4-4. ¹H NMR (DMSO-d₆) δ 12.20 (bs, 1H),8.32 (d, 1H, J=5.49 Hz), 8.26 (s, 1H), 7.13 (s, 1H), 7.03 (d, 1H, J=5.19Hz), 6.42 (bd, 1H, J=4.27 Hz), 3.52 (s, 2H), 3.29 (m, 4H), 2.51 (d, 3H,J=4.27 Hz), 2.33 (m, 4H). [M+H]+=358.1443.

[0226] Bromine (2.88 Kg, 18.0 mole) is added to a solution of3-methoxyacrylonitrile (1.50 Kg, 18.0 mole, mixture ofcis-/trans-isomers) in acetonitrile (3.00 L) at 5-10° C. The mixture isaged for 20 minutes, then pre-cooled water (about 5° C., 12.0 L) isadded and vigorous stirred for 1 hour.

[0227] NaOAc.3H₂O, (2.21 Kg, 16.2 mole, 0.90 equiv.) is added andstirred for 15 minutes and then thiourea (1.51 Kg, 19.80 mole, 1.10equiv.) is added (endothermic dissolution followed by about 10-15° C.exotherm in about 0.5 h). The mixture is aged at 15° C. for 1.5 hour,then more NaOAc.3H₂O (1.47 Kg, 0.60 equiv.) is added. It is slowlyheated to 60° C. in 1 hour and aged for 3 hours at 60° C. then cooled to10° C.

[0228] NaOH (10 N, 1.13 L, 0.625 equiv.) is added to adjust the pH to3.8-4.0. After aging for 1 hour, the product is filtered and washed withwater (11.5 L). Drying give 1.86 Kg of the crude aminothiazole as abrown solid.

[0229] The crude product is dissolved into acetone (35 L) at 50° C. andtreated with Darco KB-B (380 g) for 2 hours. It is filtered through aSolka-Floc pad and then rinsed with acetone (5 L). The filtrate isconcentrated in vacuo to about 7 L(about 5 L residue acetone). Heptane(10 L) is added in 0.5 hour and the slurry is aged for 1 hour. Theproduct is filtered and the filter cake is washed with 2/1heptane/acetone (6 L). Drying at rt affords 1.72 Kg of the aminothiazoleas a pinkish solid.

[0230] HPLC conditions: Ace-C8 4.6×250 mm column; linear gradient: 5-80%MeCN in 12 minutes, 0.1% H₃PO₄ in the aqueous mobile phase; Flow rate:1.50 ml/min; UV detection at 220 nm.

[0231] To a 1L RBF are added MTBE (500 mL), 9,9-dimethylxanthene (26.65g) and TMEDA (30.6 g). After degassing the solution, s-BuLi (155 g, 1.3M in cyclohexane) is cannulated into a dropping funnel and then slowlyadded over 30 min while maintaining the-batch temperature at 10-20° C.The mixture is then aged for 16 h at room temperature. Ph₂PCl is addedslowly-via a dropping funnel while maintain the mildly exothermicreaction at 10-20° C.

[0232] Approximately 60% of the Ph₂PCl (30 mL) is added in 0.5 hour. Themixture is aged for 15 minutes before addition of the remaining Ph₂PCl.After aged for 5.5 h at room temperature, the reaction is quenched withMeOH (2.0 mL). The product is filtered and the slightly yellow solid iswashed consecutively with MeOH (200 mL), water (200 mL), MeOH (200 mL)and MTBE (200 mL) and dried to give an off-white solid as product.

[0233] A slurry of 2-chloro-4-formylpyridine (1.49 Kg, 10.5 mole, 1.05equiv), 2-aminothiazole (1.27 Kg, 10.0 mole, 1.0 equiv), K₃PO₄ (2.34 Kg,11.0 mole, 1.1 equiv) in toluene (20 L) is degassed by twovacuum/nitrogen cycles. Pd₂(dba)₃ (114.5 g, 0.125 mmol, 2.5 mol % Pd)and Xantphos (159 g, 0.275 mole, 2.75 mol %) are then added and themixture is degassed by one vacuum/nitrogen cycle followed by bubblingnitrogen through the slurry for 10 minutes. The mixture is heated to 60°C. and degassed water (90 mL, 5.0 mole, 0.5 equiv) was added over 5minutes. The mixture is then heated to 90° C. and aged for 8 h. It iscooled to rt and filtered. The filter cake is washed with toluene (20 L)until very little DBA is observed in the wash. DMAc (24 L) is added tothe filter cake to dissolve the product. The insoluble is filtered offand washed with more DMAc (6 L). The filtrate is acidified withconcentrate HCl (110 mL) to pH 2.7. Water (3 L) is added and the mixtureis concentrated at 40-50° C. under vacuum to remove most of the residualtoluene by azeotropic distillation. More water (3×IL) is added as thedistillation progress.

[0234] The mixture is seeded and then water (13 L) is added at a rate ofabout 1.3 L/h. The product is filtered and washed with 5/4 DMAc/water(4.0 L×2), water (4.0 L), acetone (4 L×2), and then oven dried at 40Cunder vacuum (100 mmHg) with nitrogen sweep to give the product.

[0235] To a 50-L 3-neck RBF is added H₂O (6.0 L) followed by K₂CO₃ (4.56Kg) with stirring. It is cooled to 10° C. Acetonitrile (12 L) andmethylamine (40 wt % in water, 1.40 Kg) are added and the mixture iscooled to 0-5° C. Phenyl chloroformate (2.59 Kg) is then added asquickly as possible while maintaining the exothermic reaction at <15° C.1-Benzylpiperazine is added 15 min after addition of phenylchloroformate and the biphasic mixture is heated to 70° C. After agingfor 1 h at 70° C., the reaction mixture was concentrated under vacuum toremove most of the MeCN.

[0236] NaOH (7.5L 5 N NaOH) is added and the mixture is seeded. Thesuspension is then cooled to rt and aged for 1 hour. The product isfiltered and the filter cake is washed with cold NaOH (0.5 N aq, 4 L×2)and then ice-cold water (4 L×2). It is purified by recrystallizationfrom toluene (15 L) to remove any dibenzylpiperazine impurity. NaOH isused to remove phenol.

[0237] HCl (74 mL 12 N, 0.10 eq) is added to MeOH (7 L) and thenpiperazine urea 1 (2.69 Kg, 10.0 mol) is added. The mixture ishydrogenated using 5% Pd/C (180 g) under 40 psi of hydrogen pressure at40° C. for 18 h. Pd/C is slurried in MeOH (1 L) and transferred byvacuum. The SM container is rinsed with MeOH (1 L).

[0238] After confirming the completion of the reaction, the mixture isfiltered through a pad of Solka-Floc and washed with MeOH (2 L) then IPA(4 L). The colorless solution is concentrated to about 5-6 L at about40° C. under vacuum. IPA (5 L) is added followed by HCl (12 N aq, 0.767L, 0.92 eq) until the pH of the solution becomes about 3. The mixture isthen concentrated under vacuum and flushed with more IPA (5+5 L) to afinal volume of 6 L. KF of the supernatant should be <1 w% water. It isthen aged at 15° C. for 5 h.

[0239] The resulting white crystals are filtered and washed with IPA (4L). It is then dried in a vacuum oven at 40° C. with slow nitrogen sweepto give product 2.

[0240] To a slurry of the pyridine aldehyde (2.19 Kg, 94.5 w%, 9.00mole) and the piperazine urea HCl salt (1.79 Kg, 9.90 mmol) in DMAc(13.5 L) is added Et₃N (1.00 Kg, 9.90 mole) followed by acetic acid(2.16 Kg, 36.0 mole) with cooling (15° C.). After aging for 0.5 h,NaBH(OAc)₃ (2.29 Kg, 10.8 mole) is added in 8 portions (25minutes/portion).

[0241] The mixture is stirred for 1 hour and the completion of thereaction confirmed by HPLC. Water (6.8 L) is added slowly (14 h) tocomplete the crystallization. Seed with monohydrate of the free baseafter about 1-2 L of water has been added.

[0242] The product is filtered after aging for 3 hours and the filtercake washed with 3/2 DMAc/water (6.7 L), then 1/1 acetone/water (6 L)then acetone (2×4 L). Oven drying at 40 C with slow nitrogen sweepafforded the free base of Compound 4-4.

[0243] Compound 4-4(4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide) was studied as crystalline mesylate salts.

[0244] The structure of the free base of 4-4 is shown below:

[0245] The free base form of 4-4 has the molecular formula C₁₆H₁₉N₇₀Sand a molecular weight of 357.44.

[0246] Mesylate Salt of 4-4 (Forms A and B):

[0247] Crude free base of Compound 4-4 (2.70 Kg, 95.4 w%) is dissolvedinto 2/1 DMAc/THF (21.6 L) and treated with Darco G60 (675 g) at 40 Cfor 3 hours. It is filtered through a Solka-Floc pad and the filter padis washed with 2/1 DMAc/THF (4.5 L) then THF (1.8L). If there is carbonbreakthrough, then filter through a 1 micron in-line filter.

[0248] MsOH (184 g) is added and the mixture is seeded with someanhydrous mesylate salt of Compound 4-4. After stirring for 2 hours,more MsOH (542 g) is slowly added and the mixture is aged for 4 hours.The solid is filtered and washed with 2/1 DMAc/THF (6 L) then acetone(2×3 L). The wet cake is directly used to prepare the monohydrate byreslurrying in 2/1 acetone/water (5.4 L for 2 hours. More acetone (3.6)is then added and aged for 3 hours. The product is filtered and washedwith acetone (2×2.7 L) and oven dried at 40° C. and 28 inches vacuumwith nitrogen sweep afforded the mesylate salt (Form A) of Compound 4-4(3.08 Kg).

[0249] The mesylate salt of 4-4 has the molecular formula C₁₇H₂₃N₇O₄S₂and a molecular weight 453.545.

[0250] Microscopic Characteristics

[0251] Microscopic evaluation shows irregularly shaped particles ofapproximately 10-100 microns, which are birefringent under planepolarized light.

[0252] X-Ray Powder Diffraction (XRPD)

[0253] The X-ray powder diffraction pattern of the mesylate salt (FIGS.1 and 2) is indicative of crystalline material with multiple diffractionpeaks between 5° and 30° 2-theta. Form A is the monohydrate form of themesylate salt and Form B is the dehydrate, or anhydrous, form of themesylate salt.

[0254] Thermal Properties

[0255] DSC

[0256] Differential scanning calorimetry (DSC) of Form A of the mesylatesalt from 20° C. to 350° C. at a heating rate of 5° C./min showsmultiple thermal events and an endotherm at 233° C., which is attributedto melting. Form B of the mesylate salt shows an endotherm at 232° C.,at a heating rate of 5° C./min. TGA

[0257] Thermogravimetric analysis (TGA) of the mesylate salt Form A from20° C. to 400° C. at a heating rate of 10° C./min. shows a weight lossof 4.1% up to 150° C. This weight loss is attributed to the dehydrationof the monohydrate. The data suggest that the drug decomposes around300° C. TGA and DSC traces suggest that the mesylate salt Form A ismonohydrate and decomposes upon melting as determined by a sharp weightloss in the TGA at the melting temperature. The DSC and TGA confirmedthe conversion of the monohydrate to an anhydrous form (Form B). Nothermal events were observed below 200° C. for the dehydrated mesylatesalt (Form B). The dehydrate, Form B, gained about 1.2% wt. at 90-95%RH.

[0258] Hygroscopicity

[0259] The hygroscopicity of the mesylate salt was determined at 25° C.using a step isotherm program for relative humidities from 0 to 95% RHfollowing drying at 40° C./0%RH for two hours. The mesylate salt (FormA) is slightly hygroscopic at 25° C. and picks up about 4% moisture at80%RH.

[0260] The mesylate salt (Form A) was further studied using hot-stageXRPD. The mesylate salt retained the initial form at 100° C. but changedto a different form (Form B) at 175° C. Form B is stable under ambientconditions overnight. The dehydration could also be achieved bysuspending the mesylate monohydrate in hot acetonitrile. The formisolated from hot acetonitrile is identical to that obtained bythermally dehydrating the monohydrate.

[0261] Solubility

[0262] The solubility of the mesylate salt (Form A) of 4-4 at roomtemperature was determined in water, complexing agents, cosolvents andorganics that can be used in pharmaceutical processing. The results aretabulated in Table I below. TABLE I Solubility of the Mesylate Salt(Form A) in Different Organic Solvents Solvent Solubility (mg/mL) 25%HP-βCD¹ 45.0 20% HP-βCD¹ 40.7 15% HP-βCD¹ 35.3 10% HP-βCD¹ 27.1 PG 30.4PEG 1.41 Ethanol 0.98 IPA 0.64 30% PG/10% EtOH 13.6 25% PEG/10% EtOH11.6 water² 6.65

[0263] The solubility of the mesylate salt (Form A) in water is 4.7mg/mL and the pH of the resulting aqueous solution is 1.8. The initialform of the mesylate salt (Form A) is retained when it is suspended inwater for 3 weeks. A high initial solubility (>80 mg/mL) was observedfor the dehydrated mesylate salt (Form B) in water, but a whiteprecipitate was noticed upon aging the solution for two days. Therecovered solid was examined by XRPD and found to be the monohydrate(Form A).

[0264] The X-ray powder diffraction patterns for the mesylate salt(Forms A and B) are illustrated in FIGS. 1 and 2. The X-ray powderdiffraction data for these salts are summarized below in the tablesbelow:

[0265] Mesylate Salt (Form A) relatively intensity d-spacing (Å) 2-theta( ^(°) ) (%) 8.29 10.67 29.56 7.21 12.28 41.83 4.96 17.88 35.78 4.8118.45 42.25 4.19 21.21 18.35 4.14 21.49 20.98 3.92 22.68 19.56 3.8323.22 67.86 3.81 23.37 100.00 3.72 23.94 14.47 3.29 27.12 24.49 3.1128.72 15.27 2.82 31.68 14.27

[0266] Dehydrated Mesylate (Form B) relatively intensity d-spacing (Å)2-theta ( ^(°) ) (%) 9.19 9.62 44.68 6.51 13.61 19.15 5.85 15.15 19.275.65 15.69 22.18 5.43 16.33 33.50 5.35 16.58 15.85 5.00 17.75 100.004.91 18.07 34.24 4.58 19.37 90.41 4.52 19.62 48.20 4.23 21.01 25.91 4.1921.23 21.30 4.03 22.07 48.14 3.98 22.35 47.99 3.92 22.67 16.47 3.8922.87 24.48 3.73 23.83 27.97 3.56 25.02 56.79 3.53 25.23 19.75 3.2327.63 35.09 2.93 30.47 21.34

What is claimed is:
 1. A polymorphous form of a mesylate salt of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide.
 2. The polymorphic form of the mesylate salt accordingto claim 1 that is characterized by an X-ray powder diffraction patternhaving diffraction angles of: 10.67, 12.28, 17.88, 18.45, 21.21, 21.49,22.68, 23.22, 23.37, 23.94, 27.12, 28.72, and 31.68.
 3. The polymorphicform of the mesyalte salt according to claim 2 having multiplediffraction peaks between 5° and 35° 2-theta and a melting endotherm of233° C. at a rate of 5° C. per minute.
 4. The polymorphic form of themesylate salt according to claim 1 that is characterized by an X-raypowder diffraction pattern having diffraction angles of: 9.62, 13.61,15.15, 15.69, 16.33, 16.58, 17.75, 18.07, 19.37, 19.62, 21.01, 21.23,22.07, 22.35, 22.67, 22.87, 23.83, 25.05, 25.23, 27.63, and 30.47. 5.The polymorphic form of the mesylate salt of claim 4 having multiplediffraction peaks between 5° and 35° 2-theta and a melting endotherm of232° C. at a rate of 5° C. per minute.
 6. A pharmaceutical compositionthat is comprised of a polymorphous form of the mesylate salt of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide in accordance with claim 1 and a pharmaceuticallyacceptable carrier.
 7. A pharmaceutical composition that is comprised ofa polymorphous form of the mesylate salt of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide in accordance with claim 2 and a pharmaceuticallyacceptable carrier.
 8. A pharmaceutical composition that is comprised ofa polymorphous form of the-mesylate saltof-4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide in accordance with claim 4 and a pharmaceuticallyacceptable carrier.
 9. A method of treating or preventing cancer in amammal in need of such treatment which is comprised of administering tosaid mammal a therapeutically effective amount of the polymorphous formof the mesylate salt of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide in accordance with claim
 1. 10. A method of treatingcancer or preventing cancer in accordance with claim 9 wherein thecancer is selected from cancers of the brain, genitourinary tract,lymphatic system, stomach, larynx and lung.
 11. A method of treating orpreventing cancer in accordance with claim 9 wherein the cancer isselected from histiocytic lymphoma, lung adenocarcinoma, small cell lungcancers, pancreatic cancer, glioblastomas and breast carcinoma.
 12. Amethod of treating or preventing a disease in which angiogenesis isimplicated, which is comprised of administering to a mammal in need ofsuch treatment a therapeutically effective amount of the polymorphicform of the mesyalte salt of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide in accordance with claim
 1. 13. The method inaccordance with claim 14 wherein the disease is an ocular disease. 15.The method according to claim 13 wherein the ocular disease is retinalvascularization, diabetic retinopathy, age-related macular degeneration,retinal ischemia, or macular edema.
 16. A method of treating orpreventing inflammatory diseases which comprises administering to amammal in need of such treatment a therapeutically effective amount ofthe polymorphic form of the mesylate salt of4-[2-(5-cyano-thiazol-2-ylamino)-pyridin-4-ylmethyl]-piperazine-1-carboxylicacid methylamide in accordance with claim
 1. 17. A method according toclaim 16 wherein the inflammatory disease is selected from rheumatoidarthritis, psoriasis, contact dermatitis and delayed hypersensitivityreactions.
 18. A method of treating or preventing a tyrosinekinase-dependent disease or condition which comprises administering atherapeutically effective amount of the polymorphic form in accordancewith claim
 1. 19. A method of treating or preventing bone associatedpathologies selected from osteosarcoma, osteoarthritis, and ricketswhich comprises administering a therapeutically effective amount of thepolymorphic form according to claim
 1. 20. The composition of claim 6further comprising a second compound selected from: 1) an estrogenreceptor modulator, 2) an androgen receptor modulator, 3) retinoidreceptor modulator, 4) a cytotoxic agent, 5) an antiproliferative agent,6) a prenyl-protein transferase inhibitor, 7) an HMG-CoA reductaseinhibitor, 8) an HIV protease inhibitor, 9) a reverse transcriptaseinhibitor, and 10) another angiogenesis inhibitor.
 21. The compositionof claim 20, wherein the second compound is another angiogenesisinhibitor selected from the group consisting of a tyrosine kinaseinhibitor, an inhibitor of epidermal-derived growth factor, an inhibitorof fibroblast-derived growth factor, an inhibitor of platelet derivedgrowth factor, an MMP inhibitor, an integrin blocker, interferon-α,interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor,carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, and an antibody to VEGF.
 22. The composition of claim 20,wherein the second compound is an estrogen receptor modulator selectedfrom tamoxifen and raloxifene.
 23. A method of treating cancer whichcomprises administering a therapeutically effective amount of thepolymorphic form of the mesylate salt in accordance with claim 1 incombination with radiation therapy.
 24. A method of treating orpreventing cancer which comprises administering a therapeuticallyeffective amount of the polymorphic form of the mesylate salt inaccordance with claim 1 in combination with a compound selected from: 1)an estrogen receptor modulator, 2) an androgen receptor modulator, 3)retinoid receptor modulator, 4) a cytotoxic agent, 5) anantiproliferative agent, 6) a prenyl-protein transferase inhibitor, 7)an HMG-CoA reductase inhibitor, 8) an HIV protease inhibitor, 9) areverse transcriptase inhibitor, and 10) another angiogenesis inhibitor.25. A method of treating cancer which comprises administering atherapeutically effective amount of the polymorphic form of the mesylatesalt according to claim 1 in combination with radiation therapy and acompound selected from: 1) an estrogen receptor modulator, 2) anandrogen receptor modulator, 3) retinoid receptor modulator, 4) acytotoxic agent, 5) an antiproliferative agent, 6) a prenyl-proteintransferase inhibitor, 7) an HMG-CoA reductase inhibitor, 8) an HIVprotease inhibitor, 9) a reverse transcriptase inhibitor, and 10)another angiogenesis inhibitor.
 26. A method of treating or preventingcancer which comprises administering a therapeutically effective amountof the polymorphic form of the mesylate salt in accordance with claim 1and paclitaxel or trastuzumab.
 27. A method of treating or preventingcancer which comprises administering a therapeutically effective amountof the polymorphic form of the mesylate salt in accordance with claim 1and a GPIIb/IIIa antagonist.
 28. The method of claim 27 wherein theGPIIb/IIIa antagonist is tirofiban.
 29. A method of reducing orpreventing tissue damage following a cerebral ischemic event whichcomprises administering a therapeutically effective amount of thepolymorphic form of the mesylate salt in accordance with claim
 1. 30. Amethod of treating or preventing cancer which comprises administering atherapeutically effective amount of the polymorphic form of the mesylatesalt of claim 1 in combination with a COX-2 inhibitor.
 31. A method oftreating or preventing preeclampsia which comprises administering atherapeutically effective amount of the polymorphic form of the mesylatesalt of claim
 1. 32. A method of treating or preventing tissue damagedue to bacterial meningitis which comprises administering atherapeutically effective amount of the polymorphic form of the mesylatesalt of claim
 1. 33. A method to treat or prevent endometrioses whichcomprises administering a therapeutically effective amount of thepolymorphic form of the mesylate salt of claim
 1. 34. A method oftreating or preventing diabetic retinopathy which comprisesadministering a therapeutically effective amount of the polymorphic formof the mesylate salt of claim 1 in combination with a PPAR-γ agonist.